Tau
Tau is a neuronal microtubule associated protein (MAP) that is part of the fast axonal transport (FAT) system (1) . This system allows for the motor mediated movement of cargo towards (retrograde) and away from (anterograde) the cell body (1) . The system includes the motor proteins kinesin-1 and -2 and cytoplasmic dynein which walk on microtubule (MT) tracks (2) . It also includes many MAPs that help to regulate cargo transport. Tau was first isolated by the Kirshner group in the late 1970s (3) . Initally, it was thought that tau's function was to stabilize MTs since it had been purified in association with tubulin (1) . Since it's initial isolation, it as been shown that tau plays other roles in axonal transport including interacting with the actin cytoskeleton (4) , participating in signaling cascades (5) and inhibiting kinesin-1 motility (2) . There are six isoforms of tau which differ in the number of MT binding repeats (3-4) in the C-terminal and the number of acidic inserts (0-2) in the N-terminal (1) . These isoforms arise from alternate splicing of the MAPT ''gene and are expressed temporally during development (6) . The shortest isoform, 3RS tau, is the only isoform expressed in the infant brain but, in the adult brain the shortest and longest isoform (4RL tau) are expressed in a 1:1 ratio (6) . Tau in its hyperphosphorylated state is part of the neurofibrillary tangles (NFTs) that are found in the brains of Alzheimer's Disease (AD) patients (1). Because of this, it is an area of great interest for research. Conflicting hypotheses exist about the role of NFTs in AD. On one hand, NFTs are thought to be toxic and contribute to neural degeneration. On the other, NFTs are thought to be a means of protecting the neuron from apoptosis since NFTs can persist in the brain for years without causing harm. Evidence supporting both hypotheses has been presented over the years. Tau's exact role in AD remains elusive and tau itself remains a fascinating protein for study. Recombinant Tau Generating recombinant forms of tau allows for researchers to manipulate the protein not only to mimic disease states or conformational changes but also to prepare the protein for use in different imaging techniques. Phosphorylation of different residues of tau has been shown to have important implications for FAT (5) . Since tau is expressed in ''Escherichia coli ''it is not subject to post translational modifications and therefore does not get phosphorylated. To circumvent this problem, phosphomimetic forms of tau can be generated. To create a phosphomimetic, the residue of interest can be mutated to a glutamic acid or aspartic acid which provides the negative charge that phosphorylation at the site would give (5) . For single molecule experiments it is important that each protein is labeled once (7) . If the flour is attached to tau through a cysteine residue then a recombinant form of the protien must be made containing only one cysteine. Tau has two cysteine residues but one can be mutated to an isoleucine to prevent double labeling of the protein. The pET Plasmid Recombinant tau protein can be expressed using the pET vector system. This vector contains either a kanamycin or ampicilin resistance gene, a promoter, restriction enzyme sites and an origin of replication (8) . The gene of interest is under the control of the T7 promoter (8) . The T7 polymerase is encoded by the host cell which carries the gene for T7 polymerase (8) . IPTG (Isopropyl β-D-1-thiogalactopyranoside) introduction to the system induces T7 polymerase expression which in turn allows for expression of the protein of interest. BL21 (DE3) Cells BL21 (DE3) were derived from the B strain of E. coli that was first named in 1942. The (DE3) refers to the lambda DE3 phage which contains the gene for T7 polymerase under the control of the ''lac''UV5 promoter . This is needed to transcription of mRNA for the recombinant protein. Transformation of the bacteria can be done through heat shock. The bacteria are treated with 2-mercaptoethanol and the vector DNA is added to the culture prior to heat shock which allows for the entry of the DNA into the cells. Following immediate incubation on ice, the cells are incubated for an hour at 37 degrees Celcius and plated on LB agar. A small overnight culture is made using the transformed cells. The overnight culture can then be used to innoculate the expression culture. Once the expression culture has reached log phase, expression of the recombinant protein can be induced by addition of IPTG. Protein Purification Once the tau construct has been expressed, the cells are lysed in cold lysis buffer, the mixture is sonicated and the lysates are centrifuged. The supernanant is then collected and boiled to denuture unwanted proteins. The boiled supernatant is centrifuged again. This new supernatant is passed through a 0.22 um filter in preparation for ion exchange chromatography. The filtered supernatant is then put through an anion exchange column which binds any unwanted protein. The flow through from this column is collected and passed through a cation exchange column. Because of tau's overall positive charge, it will bind to this column from which it can be eluted with an increasing salt concentration. Dialysis against a buffer can then be done to remove excess salt from the protein. References 1. Ballatore C, Lee VM, & Trojanowski JQ (2007) Tau-mediated neurodegeneration in Alzheimer's disease and related disorders. ''Nature reviews. Neuroscience 8(9):663-672. 2. Dixit R, Ross JL, Goldman YE, & Holzbaur EL (2008) Differential regulation of dynein and kinesin motor proteins by tau. Science 319(5866):1086-1089. 3. Weingarten MD, Lockwood AH, Hwo SY, & Kirschner MW (1975) A protein factor essential for microtubule assembly. Proceedings of the National Academy of Sciences of the United States of America 72(5):1858-1862. 4. He HJ'', et al.'' (2009) The proline-rich domain of tau plays a role in interactions with actin. BMC cell biology 10:81. 5. Andreadis A (2005) Tau gene alternative splicing: expression patterns, regulation and modulation of function in normal brain and neurodegenerative diseases. Biochimica et biophysica acta 1739(2-3):91-103. 6. Kanaan NM'', et al.'' (2012) Phosphorylation in the amino terminus of tau prevents inhibition of anterograde axonal transport. Neurobiology of aging 33(4):826 e815-830. 7. McVicker DP, Chrin LR, & Berger CL (2011) The nucleotide-binding state of microtubules modulates kinesin processivity and the ability of Tau to inhibit kinesin-mediated transport. The Journal of biological chemistry 286(50):42873-42880. 8. Samuelson J (2011) Bacterial Systems. Production of Membrane Proteins, (Wiley-VCH Verlag GmbH & Co. KGaA), pp 11-35.